Summary Exercise robustly enhances cognitive performance across the lifespan but mechanisms are not well understood. The long-term goal of this research program is to elucidate the neurological mechanisms by which exercise improves cognition. The objective of this application is to use an in vitro model to identify factors released from contracting primary muscle fibers that increase the connectivity and synchronous activation of primary hippocampal neuron cultures. A recent line of investigation has presented muscle-released circulating factors produced during physical activity as potential causal agents driving changes in hippocampal plasticity. However, the full complement of factors, including exosomes, which might contribute to long distance communication between muscles and hippocampal neurons has not been well characterized. Identifying the factors would be useful for therapeutic applications aiming to recapitulate effects of exercise for neuronal regeneration and repair. The central hypothesis is that muscle fiber contractions release factors which have the capability to enhance the rate of maturation of hippocampal neuronal circuits. The hypothesis is supported by preliminary studies showing neuronal cultures exposed to the media from contracting muscle fibers display more rapid maturation of neuronal connections and synchronous activation patterns as compared to neuronal cultures exposed to control media. One of the PIs has a productive research program on exercise-brain interactions, and the other on mechanical micro-environment effects on cell functionality. The PIs have developed multiple innovative methods for powerful hypothesis testing and exploration. The objectives of this application will be accomplished by pursuing 3 specific aims: 1) Determine the extent to which factors released from primary skeletal muscle cells subjected to prescribed range of contraction regimens accelerate synchronous firing of cultured primary hippocampal neurons. 2) Identify novel compounds released from contracting muscle fibers and explore whether exosomes are also released. 3) Determine the extent to which cross-talk between hippocampal neurons and contracting muscle cells affects maturation and connectivity of cultured hippocampal neuronal circuits. A novel platform which allows cross-talk between hippocampal neurons and muscle-motor neuron units but prevents physical contact will be used. State-of-the-art peptidomics methods will lead to the discovery of new molecules released by contracting muscle fibers that influence plasticity of neurons, and new imaging methods will be used to explore whether exosomes are also released. Elucidating and unequivocally establishing mechanisms underlying pro-cognitive effects of exercise holds the key to discover novel and more efficient ways to maintain, promote and improve cognitive performance. The proposed research is highly innovative, it addresses pressing questions in the field using very novel strategies and state-of-art technologies that will allow us to generate causal, mechanistic data on how muscles communicate with hippocampal neurons.